Various environmental and industrial chemicals can perturb male reproductive function. The objectives of these studies are to define subcellular target sites in testicular cells in primary culture. For FY91, efforts have continued to define the mechanism of action of mono-(2-ethylhexyl)-phthalate on Sertoli cells utilizing fluorescent probes to characterize effects on subcellular targets. For saligenin, cyclic-O-tolyl phosphate, efforts have focused on assembling and optimizing methods for assessing protein phosphorylation. A small format gel system was tried and proved unworkable: large format gels are being evaluated now. Germ cells are an often overlooked target of testicular toxicants. A subset of these studies is designed to evaluate 1) methods of Isolating and maintaining germ cells: 2) methods of labelling germ cell (and Sertoli cell) DNA and RNA; and 3) appropriate endpoints of germ cell function (genome activation). Finally, pilot studies are underway to test the feasibility of isolating stages of living seminiferous epithelium, and maintain these fragments in culture. Toxicants will be applied, and variations in responses assessed. Thus, studies this year have broadened into non-somatic testicular cell types in an attempt to more completely evaluate potential mechanisms.